1. Field of Invention
The present invention relates generally to the fields of molecular biology, pharmacology and to gene therapy. More particularly, it concerns methods and compositions comprising ferritin-H for regulation of genes related to iron metabolism. and regulation, and for treatment of various diseases, including neurodegenerative diseases and neuromuscular diseases.
2. Background of the Invention
Hemoglobin comprises four protein chains, two alpha chains and two beta chains (α2β2), interwoven together, each with its own molecule of iron and with a combined molecular weight of about 68 kD. The hemoglobin macromolecule is normally glycosylated and upon absorbing oxygen from the lungs transforms into oxyhemoglobin (HbO2). There are at least six distinct forms of hemoglobin, each expressed at various times during development. Hemoglobin in the embryo is found in at least three forms, Hb-Gower 1 (ζ2ε2), Hb-Gower 2 (α2ε2), and Hb-Portand (ζ2γ2). Hemoglobin in the fetus comprises nearly totally HbF (α2γ2), whereas hemoglobin in the adult contains about 96% HbA (α2β2), about 3% HbA2 (α2δ2) and about 1% fetal HbF (α2γ2). The embryonic switch of globin expression from ζ- to α- and from ε- to γ- begins in the yolk sac. However, chains of embryonic ζ- and ε- have been found in the fetal liver and complete transition to the fetal form does not occur until late in fetal development. The fetal switch from γ- to β- begins later in erythropoiesis with the amount of β-globin produced increasing throughout gestation. At birth, β-globin accounts for about 40% of non-α-globin chain synthesis and thereafter continues to rapidly increase.
Defects or mutations in globin chain expression are common. Some of these genetic mutations pose no adverse or only minor consequences to the person; however, most mutations prevent the formation of an intact or normal hemoglobin molecule through a functional or structural inability to effectively bind iron, an inability of the chains or chain pairs to effectively or properly interact, an inability of the molecule to absorb or release oxygen, a failure to express sufficient quantities of one or more globin chains or a combination of these malfunctions. For example, substitution of valine for glutamic acid at the sixth position of the β chain produces HbS hemoglobin and was found to occur in about 30% of black Americans. In the HbS heterozygote, only about 40% of total hemoglobin is HbS with the remainder being the more normal HbA.
In studies of hemoglobin, “Hb” refers to hemoglobin. HbA refers to normal adult hemoglobin, HbF refers to fetal hemoglobin, and HbS refers to sickling hemoglobin.
Background for Sickle Cell Disease:
Upon deoxygenation, HbS hemoglobin molecules undergo aggregation and polymerization ultimately leading to a morphological distortion of the red cells which acquire a sickle or holly-leaf shape. Sickling has two major consequences, a chronic hemolytic anemia and an occlusion of small blood vessels that result in ischemic damage to tissues. Further, when exposed to low oxygen tensions, polymerization converts HbS hemoglobin from a free-flowing liquid to a viscous gel. Consequently, the degree of pathology associated with sickle cell anemia can be correlated with the relative amount of HbS in the patient's system.
Individuals with severe sickle cell anemia develop no symptoms until about five to six months after birth. In these infants it was determined that fetal hemoglobin did not interact with HbS hemoglobin and, as long as sufficient quantities were present, could modulate the effects of HbS disease. This modulating effect of γ-globin is also observed with other β-globin disorders, such as HbC and HbD, and other mutations of the β chain. HbS polymerization is also significantly affected by the hemoglobin concentration of the cell. The higher the HbS hemoglobin concentration, the greater the chances for contact between two or more HbS hemoglobin molecules. Dehydration increases hemoglobin concentration and greatly facilitates sickling.
The average sickled red blood cell survives for about 20 days or less in the body, as compared to the 120-day life span of a normal red blood cell. Individuals with HbS syndromes have frequent infections, chronic hemolysis with a striking reticulocytosis and hyperbilirubinemia. The course of the disease is typically punctuated with a variety of painful crises called vaso-occlusive crises. These crises represent episodes of hypoxic injury and infarction in the organs, abdomen, chest, extremities or joints. Leg ulcers are an additional manifestation of the vaso-occlusive tendency of this disease. Central nervous system involvement is common producing seizures and even strokes. Aplastic crises, also common, represent a temporary cessation of bone marrow activity and may be triggered by infections, folic acid deficiency or both. Crises are episodic and reversible, but may be fatal. Damage from crisis episodes tends to be cumulative and even in those individuals with milder forms of sickle cell disorder, life-spans can be greatly reduced.
Many current research and experimental treatment efforts are aimed at the processes that cause red blood cells to sickle. Hydroxyurea has been found to stimulate the production of fetal hemoglobin, a type of hemoglobin found in the fetus and small infants, which is able to block the sickling action of red blood cells. Human Genome Research Institute indicates that those treated with hydroxyurea need fewer blood transfusions and have fewer attacks of acute chest syndrome. The University of Maryland states that it is “currently the only agent in general use to prevent acute sickle-cell crises” but has no effect on 25 percent of patients and cannot be used during pregnancy.
Some current research efforts to treat sickle cell disease involve correcting the defective hemoglobin gene and inserting it into the bone marrow of those with sickle cell to stimulate production of normal hemoglobin. For example, researchers from Harvard Medical School and MIT, with support from the National Institutes of Health, were able to correct sickle cell disease in mice using this approach in 2001. Researchers used bioengineering to create mice with a human gene that produces the defective hemoglobin causing sickle cell disease. Bone marrow containing the defective hemoglobin gene was removed from the mice and genetically “corrected” by the addition of the anti-sickling human β-hemoglobin gene. The corrected marrow was then transplanted into other mice with sickle cell disease. The genetically corrected mice began producing high levels of normal red blood cells and showed a dramatic reduction in sickled cells. Scientists are hopeful that the techniques can be applied to human gene transplantation using autologous transplantation, in which some of the patient's own bone marrow cells would be removed and genetically corrected. However, additional research and development is required before this gene therapy approach is applicable in human. Furthermore, this approach can only reach a very small number of patients because of the high cost and that the technological complexities involved in gene therapy and stem cell (bone marrow cell, cord blood stem cell, etc) transplant require that these procedures are performed in a major health research center.
Background for Thalassemia:
The thalassemia syndromes are a heterogenous group of disorders all characterized by a lack of or a decreased synthesis of the globin chains of HbA. Deficiencies of β-globin expression are referred to as β-thalassemias and deficiencies of αZglobin, α-thalassemias. The hemolytic consequences of deficient globin chain synthesis result from decreased synthesis of one chain and also an excess of the complementary chain. Free chains tend to aggregate into insoluble inclusions within erythrocytes causing premature destruction of maturing erythrocytes and their precursors, ineffective erythropoiesis, and the hemolysis of mature red blood cells. The underlying defects of hemoglobin synthesis have been elucidated over the years and largely reside in the nucleic acid sequences which express or control the expression of α- or β-globin protein.
Mammalian globin gene expression is highly regulated during development. The human beta-globin gene cluster includes one embryonic gene, epsilon (ε), two adult beta globin genes, beta (β) and delta (δ), two fetal beta globin genes G-gamma (G-γ) and A-gamma (A-γ), which differ by only one amino acid, and at least one pseudo-beta gene, psi beta (ψβ). All are expressed from a single 43 kilobase segment of human chromosome 11. Fetal beta type globin, or γ-globin, is expressed in the earliest stages of mammalian development and persists until about 32 to 34 weeks of gestation. At this stage, the adult forms of β-globin begin to be expressed and substitute for the fetal proteins.
Each β-globin gene comprises three exons which encode about 146 amino acids, two introns and a 5′-untranslated region containing the promoter sequences. Biosynthesis of β-globin begins with transcription of the entire gene followed with RNA processing of the message, removal of the introns by splicing, poly-A addition, capping and post-transcriptional modifications. The mature mRNA molecule is exported from the nucleus and translated into β-globin. Defects in each of these functions have been found associated with specific thalassemias. Identified mutations include single-nucleotide deletions, insertions and substitutions, frame shift mutations, deletions of entire segments of coding or controlling regions, improper termination signals, aberrant splicing signals, and multiple mutations. β°-thalassemias are characterized by a complete absence of any β-globin chains; β+-thalassemias are characterized by a detectable presence of a reduced amount of β chains.
There are three principal categories of β-thalassemia, thalassemia major, thalassemia intermedia and thalassemia minor. Patients with thalassemia minor may be totally asymptomatic and are genotypically β+/β or β°/β. Although red cell abnormalities can be detected, symptoms are mild. Thalassemia intermedia patients are most often genotypically β+/β+ or β°/β and present severe symptoms which can be alleviated with infrequent blood transfusions. In contrast, thalassemia major patients are genotypically β°/β°, β°/β+ or β+/β+, and require regular and frequent transfusions. Children suffer from severe growth retardation and die at an early age from the profound effects of anemia. Those that survive longer suffer from morphological changes. The face becomes distorted due to expansion of marrow within the bones of the skull, hepatosplenomegaly ensues, there is a delayed development of the endocrine organs including the sexual organs, and a progressive iron overload with secondary hemochromatosis.
There are two direct consequences of β-thalassemia. First, there is an inadequate formation of HbA and, therefore, an impaired ability to transport oxygen. There are also multiple effects attributable to an imbalance between α- and β-chain syntheses. Surprisingly, the pathological consequences of globin chain imbalance appear to be the more severe. Free α chains form unstable aggregates that precipitate within red cell precursors in the form of insoluble inclusions. These inclusions damage cellular membranes resulting in a loss of potassium. The cumulative effect of these inclusions on the red blood cells is an ineffective erythropoiesis. An estimated 70% to 85% of normoblasts in the marrow are eventually destroyed. Those that do escape immediate destruction are at increased risk of elimination by the spleen where macrophages remove abnormal cells. Further, hemolysis triggers an increased expression of erythropoietin which expands populations of erythroid precursors within bone marrow and leads to skeletal abnormalities. Another severe complication of β-thalassemia is that patients tend to have an increased ability to absorb dietary iron. As most treatments for thalassemia involve multiple transfusions of red blood cells, patients often have a severe state of iron overload damaging all of the organs and particularly the liver. To reduce the amount of iron in their systems, iron chelators are typically administered. Although helpful, patients succumb at an average of between about 17 to 35 years of age to the cumulative effects of the disease and iron overload.
Genotypic variations in healthy individuals have been identified wherein adult βZglobin is not formed, but severe complications are avoided. These patients constituitively express fetal or γ-globin protein in amounts sufficient to substitute for the missing β-globin protein. This hereditary persistence of fetal hemoglobin (HPFH) involves one or both of the fetal beta globin genes, A-γ and G-γ. Apparently, consistent production of either γ-globin protein accomplishes the necessary functions of the abnormal or missing β-globin protein.
Background for Skin Cancer and Other Cancers:
Ultraviolet (UV) light is known to be damaging to human skin and has been implicated in the etiology of skin cancers. Recent studies have revealed that ferritin is elevated in cultured skin cells exposed to UV light, and it has been postulated that the increased ferritin represents the skin cell's attempt to protect itself from free radical damage by binding and sequestering iron which could, in turn, cause oxidative and free radical-mediated damage.
The rationale for other cancers is similar. Iron has been implicated as an etiologic agent in skin cancer, hepatomas (liver cancer), renal cell carcinoma (kidney cancer), neuroblastomas, leukemias, and breast cancer. Ferritin-H, an iron chelator, is protective against carcinogenic events in cells that give rise to all of these cancers. When human skin is treated in such a way as to transfect them with a ferritin-H subfamily peptide or gene that expresses the peptide, protection from UV-induced damage is provided to the cells. Ferritin-H-subfamily peptides are thought to be superior in this regard since they can sequester iron and not release it readily and can do so without altering normal aspects of the cells iron metabolism and other functions. Ferritin-L-subfamily peptides, on the other hand, are likely to cause even more harm in that they readily give up iron which exacerbates the problem by increasing free iron and radical generation. Thus, delivering a ferritin-H-subfamily peptide or a gene (expression clone) for the peptide to the target cells is protective and/or corrective of events that lead to cancer. Similarly, agents that activate the endogenous ferritin-H-subfamily gene or genes are also beneficial.
It is realized that all human ferritins, even those highly enriched in ferritin-L, require a small amount of ferritin-H and its associated ferrooxidase activity to carry out the functions of iron storage and release. It is the balance between ferritins L and H that is critical. Increasing the balance in favor of ferritin-H, even to the point of great excess of ferritin-H, appears to mediate a cell's return to healthy iron management.
Background for Neurodegenerative Diseases:
The distribution of free iron and of ferritin both change during brain development in animals and humans. Increased iron is found in the basal ganglia, beginning early in the disease process, in both Parkinson's disease and Huntington's disease. There is an increase in iron in several areas of the brain in Alzheimer's disease, in other dementias, and in aging; and the distribution of isoferritins in a variety of brain areas is different and changes in the above diseases. Ferritin-H, but not ferritin-L, is present in the nucleus of neuronal cells in the cortex of developing rat brains and is protective against oxidative damage that is caused by free iron. Rationale: Ferritin-H decreases in critical brain cells during aging and neurodenerative diseases, whereas free iron and iron released from localized ferritin-L are implicated in oxidative damage in diseases and dementia. Ferritin-H or a related subfamily peptide is protective against a variety of neurodegenerative changes associated with aging, the above diseases and dementias. Likewise, an expression clone of a ferritin-H-subfamily gene and/or a regulator of ferritin-H-subfamily genes, if delivered to the appropriate brain area and to specific cells, is predicted to be protective.
Background for Friedreich's Ataxia and Related Neuromuscular Disorders:
Deletion of YDL120, the yeast homologue of the human gene responsible for Friedreich's ataxia, elicits decreased cellular respiration associated with decreased cytochrome c oxidase activity and, in certain nuclear backgrounds, mitochondrial DNA is lost. In the null mutants, the cellular growth is highly sensitive to oxidants, such as H2O2, iron and copper; and ferrous sulfate elicits loss of mitochondrial DNA. Mitochondria of the null mutants contain ten times more iron than wild-type. The neurodegeneration observed in Friedreich's ataxia can be well explained on the basis of a mitochondrial iron overload responsible for an increased production of highly toxic free radicals. Rationale: Since iron accumulation is implicated in the etiology of Friedreich's ataxia, both the initial appearance of symptoms and the progression of this disease are slowed or halted by sequestering the free iron. Transfection of ferritin-H-subfamily peptides or expression clones and/or treatment with agents that up-regulate expression of the endogenous ferritin-H-subfamily genes are ameliorative.
Background for Atherosclerosis:
Strong epidemiological evidence is available that iron (i.e., iron excess) is an important factor in the process of atherosclerosis and that iron depletion has cardiovascular benefits and protects against ischemic heart disease. Iron-catalyzed generation of free radicals contributes to vessel wall damage, to plaque formation and, by both mechanisms, to cardiac vessel damage. Once again, intracellular iron release from ferritin-L is implicated as a source of the iron contributing to this etiology; ferritin-H is protective by chelating and sequestering the free and released iron. Rationale: Transfecting the appropriate cell with a ferritin-H subfamily peptide or gene expression clone or with a gene regulator that activates the endogenous ferritin-H subfamily gene(s) in artery wall cells or cellular elements of atherosclerotic plaques prevents or reverses artery blockage.
What is needed is an elucidation of the mechanism of developmental hemoglobin (Hb) switching which allows the reactivation of fetal Hb in adult humans, a manipulation that alleviates the clinical manifestations of sickle cell disease, β-thalassemia, and related diseases. Inactivation of the mutated form of the adult β-globin gene that causes sickle cell disease is also of clinical value, since it results in a compensatory increase in fetal γ-globin expression.